TASK-2 is a member of the two-pore domain K⁺ channel (K_2P) family and is sensitive to changes in extracellular pH; currents are maximal at alkaline pH but are progressively inhibited as the pH decreases (Reyes et al. 1998). TASK-2 is located principally in epithelial tissues, and is present in all nephron segments (Morton et al. 2002). TASK-2 has high sequence homology with TASK-4, yet lower similarity to TASK-1 and -3. In TASK-1 and -3, mutation of a charged amino acid, histidine, at position 98 abolished the pH sensitivity of this channel (Kim et al. 2000; Morton et al. 2003). However, no such homologous residue is present in TASK-2 implying a different pH sensing mechanism. pH sensing must involve titration of charged residues, and thus we have examined the involvement of charged residues in the extracellular domain of TASK-2 between the first transmembrane segment and pore region by mutation to the neutral amino acids glutamine (Q) and asparagine (N).

Wild-type (WT) murine TASK-2 cDNA was subcloned into the bicistronic vector pIRES-CD8 (Invitrogen). Single point mutations were generated by a PCR-based mutagenesis approach and confirmed by automated fluorescence sequencing (Lark). Chinese hamster ovary (CHO) cells were transfected with either WT or mutant plasmid using Fugene transfection reagent (Roche). Twenty-four to 72 h post-transfection CHO cells expressing the CD8 antigen were positively identified by incubation with immunomagnetic particles coated with anti-CD8 antibody (Dynal) and subjected to whole-cell patch clamp analysis. Whole-cell currents were recorded in mammalian Ringer solution containing (mM): NaCl 145, KCl 5, MgCl₂ 1, CaCl₂ 2, Hepes 5, Pipes 5, titrated to between pH 5.8 and 8.8 with KOH or HCl. Currents were normalized with respect to those at pH 8.8 and K_d values calculated with a single binding site model. Results are given as means ± S.E.M. and statistical significance tested using Student’s unpaired t test.

The K_d for WT channels was pH 7.47 ± 0.08 (n = 9). The K_d values of mutant channels E28Q, K32N, K35N and K47N were all significantly lower than WT (P < 0.05); 7.12 ± 0.09 (n = 9), 7.20 ± 0.06 (n = 9), 7.27 ± 0.04 (n = 14) and 7.24 ± 0.06 (n = 12) respectively. The decrease in K_d implies a reduction in the pH sensitivity of the mutant channel. The K_d value for mutant channel H44N was not significantly different from WT; 7.43 ± 0.08 (n = 11).

The large extracellular domain of TASK-2 comprises approximately 50 amino acids, 13 of which are charged and therefore titratable by protons. Amongst these charged residues are five that are unique to TASK-2. The mutation of four of these charged residues to neutral amino acids resulted in a decrease in pH sensitivity. The exact mechanism of this reduction is unknown. The residues may themselves be titrated by protons or contribute to the effective concentration of protons at the pH sensor. In conclusion, three charged amino acids in the large extracellular domain contribute to the pH sensitivity of TASK-2.

This work was supported by the Wellcome Trust.